The development of vaccines based on small antigenic epitopes is hampered by the inability of the small antigen to elicit a good immune response in a host animal. The use of carrier immunogens provides some assistance in the immune response, but often decreases the specific activity and yield of the response against the desired antigen. Methods for conjugation of antigens to carrier agents are costly, and generally utilize hazardous chemicals. Covalent coupling of antigen to a carrier protein is inherently variable, resulting in an antigen with an imprecise structure, compromising vaccine potency. The use of adjuvants also tends to decrease the yield of specific antibodies and can be harmful to the animal host, causing abcesses, skin lesions, and hypersensitivity. These factors are unacceptable for the production of a commercially useful vaccine.
Chimeric molecules formed of large carrier proteins with attached peptide epitopes have been suggested as useful vaccines for small peptide antigens. However, added peptides extending from a three-dimensional protein are generally susceptible to proteolytic degradation. Insertion of an antigenic peptide into an interior portion of a carrier protein may avoid degradation problems, but disruption of the carrier protein's native sequence can alter the carrier's three dimensional structure and thus its function, including its ability to act as an efficient immunogen.
The non-toxic beta subunit of cholera toxin (CTB) and the related B subunit of heat-labile enterotoxin from E. coli (LTB) are powerful immunogens that have been suggested for use as carriers of foreign epitopes. In studies testing the activity of CTB, antigenic peptides have been genetically fused to either the N- or C-terminus and tested for activity. These constructs were generally susceptible to rapid proteolytic degradation of the terminally fused peptide. (European Patent Application No: 89312713.4, published Jun. 13, 1990.) In other studies, a CTB-peptide molecule having a 10 amino acid peptide from the HIV-1 gp120 envelope protein substituted for eight amino acids in CTB at positions 56-63 was shown to be resistant from proteolytic degradation as compared with an N-tenninal CTB-peptide product. However, only a detectable response to the substituted gp120 epitope was obtained, and only in some, not all animal hosts. (Backstrom, et al., 1994, Gene 149:211-217.)
It is therefore highly desirable to develop an efficient and commercially useful process for producing immunogenic molecules containing antigenic peptide epitopes for use as vaccines, where the immunogenic molecule permits good recognition of the epitope as antigenic without high susceptibility to proteolytic degradation and produces a good immune response against the inserted antigen when administered to a host animal in the absence of adjuvant.